PROJECT SUMMARY: The circadian system is an important network hub coordinating cellular functions and homeostasis. Age-related alterations in the human circadian system are implicated in Alzheimer?s and other neuronal pathologies. Recent evidence in fruit flies and mice suggests correlation between disrupted rhythms and neurodegeneration; however, very little is known about mechanisms involved. To investigate these mechanisms, we compared circadian transcriptome in heads of young and old Drosophila using RNA-seq. We found that several genes, early-life cyclers (ELCs), that were expressed in young flies in a rhythmic fashion lose cycling pattern to become constitutively low or high in old flies. We also uncovered a group of genes, which we termed late life cyclers (LLCs), that were low and arrhythmic in heads of young flies but became strongly rhythmic in heads of old flies. This group contains known stress- responsive genes that are induced in young flies in response to oxidative stress or hyperoxia. Based on these findings from our recently published data, we hypothesize that the circadian system is rewired during aging through a combination of alterations in inputs from, and outputs to, stress-response pathways, and changes in post-transcriptional regulation by age-altered microRNA expression. Because of the connections between the circadian system and neurodegeneration, we expect some of these changes could be harmful for neuronal heath, and others could be part of a protective mechanism. In Aim 1, we will measure genome-wide binding of the core circadian transcription factors (TFs) CLK and CYC, stress responsive TFs, and RNA Polymerase II through ChIP-Seq and identify age-specific binding events that could be responsible for these regulatory changes. In addition, we will perform ATAC-seq to measure chromatin accessibility. In Aim 2, we will develop network models of gene regulation by combining this new data, along with our existing RNA-seq data and forthcoming small RNA-seq data. We will build computational models and analyze genomic data to create a mechanistic understanding of the epigenetic changes leading to the observed age-onset changes in diurnal expression patterns. By comparing the networks that we will build for young and old flies, we will be able to identify candidate regulators of aging, neurodegeneration (CRANs) that we will follow up on. In Aim 3, we will study the role of these CRANs in neuronal health, lifespan, and behavioral rhythms. We will use genetic manipulation to determine the causative gene regulatory events responsible for changes in health and neurodegeneration. The proposed work should reveal clock-controlled pathways that protect the brain from age-related damage, as well as examples of age-onset dysregulation of the clock network or connected pathways. Given the conserved molecular basis of circadian clock and aging biology, we expect these pathways will also function in humans. !